Fig 6. PLP-1 activity does not affect defective Ca²⁺ dynamics upon loss of PKAc1.

(A) Live cell imaging of PKAc1 cKD/Δplp1 without and (B) with ATc. (C) PKAc1 cKD treated with ATc and 50 μM DCCD. All parasite lines are stably expressing GCaMP6/mCherry at the uprt locus. In A, B, and C, (i) shows images of the individual representative movie, showing stills when parasites are extracellular, invading, and upon completion of invasion (blue arrow) and then when the normalised GCaMP/mCherry ratio reaches 35% of maximum (see S10, S11 and S12 Movies, respectively). (ii) Shows, in each case, a graphical representation of tracked parasites in (i), where blue arrow denotes the moment of completion of invasion and green arrow shows the moment of host cell egress. Dotted line marks 35% of maximum GCaMP/mCherry ratio, defined as ‘baseline’. (iii) Shows, in each case, overlays of all traced parasites. S9 Fig shows individual traces of each tracked parasite for PKAc1 cKD/Δplp1, with and without ATc, and S10 Fig outlines individual traces of PKAc1 cKD + ATc + 50 uM DCCD. (D) Graphical representation of each tracked parasite across all conditions, showing time to reach 35% of maximum, which is defined as ‘baseline’. Parental and PKAc1 cKD lines are from Fig 5. (E) Graphical representation of normalised GCaMP6/mCherry ratio at t = 100 seconds across all parasites in all conditions. Data in D and E are represented as mean ± 95% confidence interval. P values are calculated pairwise, using an unpaired two-tailed t test, where **** ≤ 0.0001. Individual numerical values underlying (D) and (E) may be found in S1 Data. ATc, anhydrotetracycline; cKD, conditional knockdown; DCCD, N,N′-Dicyclohexylcarbodiimide; GCaMP, GFP-Calmodulin-M13-peptide-6; PKAc1, protein kinase A catalytic subunit 1; PLP-1, perforin-like protein 1.